Project Summary/Abstract Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is one of the deadliest pathogens on the planet, and for decades, TB has been the leading cause of death due to infectious disease. The cell envelope of Mtb forms a notoriously tough barrier around the cell, protecting the bacterium from harsh agents in the environment such as antibiotics and host immune responses. At the same time, Mtb imports nutrients from the host cell, such as cholesterol, across the cell envelope. Transport of lipids, metabolites and nutrients across the cell envelope, between the inner and outer membranes is critical for building and maintaining the cell envelope itself, and for import of key factors required for bacterial growth. Therefore, the transport systems that facilitate this trafficking are critical for allowing Mtb to survive and thrive in its intracellular niche, most typically macrophages in the lungs. The MCE (Mammalian Cell Entry) family of proteins are transport systems that have been implicated as virulence factors in Mtb, and are an expanded protein family in mycobacteria compared with other double-membraned bacteria. In Mtb, several lines of evidence suggest that MCE systems are important for importing nutrients such as cholesterol and fatty acids. Recent work on E. coli MCE systems has shown that these are multi-protein complexes anchored in the inner membrane of double-membraned bacteria, and may play an important role in the maintenance of outer membrane integrity, raising the possibility that this may also be a role that MCE proteins play in Mtb. The structure and mechanisms of the highly complex MCE systems in Mtb remain unknown, and studying these is critical for understanding how MCE systems work, what their substrates are, and how they may be linked to virulence. This proposal is focused on biochemical and structural characterization of MCE transport systems from Mtb and the non-pathogenic model, Mycobacterium smegmatis. Using single particle cryo-EM, mass spectrometry, biochemical and functional assays, we aim to study the structure and function of endogenous Mtb MCE systems, decipher which protein subunits come together to form complexes and define protein-protein interactions that are important for the systems to assemble and function. The results of this work will provide important insights into the structure and function of the large, multi-protein MCE complexes in the Mtb cell envelope, and how they influence replication and virulence in the host.